Arne Bøyum

The Effect of Strenuous Exercise, Calorie Deficiency and Sleep Deprivation on White Blood Cells, Plasma Immunoglobulins and Cytokines

Moderate exercise appears to stimulate the immune system, but there is good evidence that intense exercise can cause immune deficiency. In the present study the authors examined the effect of continuous physical exercise (35% of VO2 max), calorie deficiency and sleep deprivation on the immune system of young men participating in a 5–7 days military training course. There was a two–three fold increase of neutrophils from day 1, the values remained high and decreased slightly at the end of the course. Monocyte counts also increased with a pattern similar to that of neutrophils. Eosinophils decreased to 30% of control and lymphocyte numbers decreased by 30–40%. All the major subgroups (CD4 T cells , CD8 T cells, B cells, NK cells) were reduced. Neutrophil function, as tested by measuring chemotaxis, was significantly stimulated during the first days of the course, in particular in the group with the lowest calorie intake. The mitogenic response of lymphocytes to PHA and Con A was variable, ranging from stimulation during one course to no effect in another course. Serum levels of immunoglobulins decreased significantly during the course. IgG was reduced by 6–7%, IgA by 10–20% and IgM by 20–35%. The authors found no changes of interleukin 1, 2 and 4 during the course, but a (12–20%) reduction (P<0.01) of interleukin 6 , and an increase (P<0.01) of granulocyte–macrophage colony stimulating factor. Altogether the results from the ranger course present a mixed‐up picture. The non‐specific phagocyte‐related immunity was enhanced. On the other hand, the data indicate that even a moderate physical activity, around the clock, caused significant suppression of a number of parameters reflecting the status of the specific, lymphocyte‐related immunity. It is noteworthy, however, that there was no significantly increased infection rate during the course or in the first 4–5 weeks thereafter.

Granulocyte chemiluminescence response to serum opsonized zymosan particles ex vivo during long-term strenuous exercise, energy and sleep deprivation in humans

The chemiluminescence response of granulocytes to serum opsonized zymosan particles (SOZ) ex vivo was investigated during two ranger training courses lasting 7 days with continuous moderate physical activities corresponding to about 32% of maximal oxygen uptake or 35 000 kJ · 24 h−1, with energy deficiency (energy supply 0-4000 kJ · 24 h−1), and less than 3-h sleep during the 7 days. Significant granulocytosis in combination with a lymphopenia in peripheral blood was observed during the whole course. A priming of the granulocytes for accentuated chemiluminescence response to SOZ was observed during the first days of the course with a maximal increase on day 3 in course A (+35% of control response) and on day 1 in course B (+ 12%). Thereafter, reduced responses to SOZ compared to control values (−28% and −21% in course A and B) were observed. In course A, a group (n = 8) receiving 5000 kJ · 24 h−1 of additional energy, showed a more pronounced priming (maximum +57% versus +21 % of control response) during the first days. In course B, all the cadets had 3 h of organised rest/sleep on day 5, and a second priming of the chemiluminescence response was observed on the subsequent 2 days. These data indicated that moderate, continuous, predominantly aerobic physical activities for 1–3 days around the clock primed the production of reactive oxygen species in granulocytes. This priming may be beneficial for, for example, host defence against micro-organisms, but may also contribute to inflammatory damage to normal tissues such as muscle, tendons and joints during exercise. However, when the moderate exercise continued for several more days, a down-modulation of the granulocyte response was observed. The findings of this study further support the possibility that moderate physical activity stimulates immunity, while more extreme duration of the same activities may result in a down-modulation of nonspecific (and specific) immunity.

Effect of VIP on the respiratory burst in human monocytes ex vivo during prolonged strain and energy deficiency

VIP-stimulated cyclic AMP production and VIP effect on the production of reactive oxygen compounds in human monocytes activated by serum opsonized zymosan (respiratory burst) were studied during a ranger training course lasting for five days with almost continuous physical activity, and deficiency of sleep and energy. Respiratory burst was inhibited and cyclic AMP production was stimulated by VIP on all days. Maximum cyclic AMP production stimulated by VIP (0.1 microM) on the day of control was 148.6% of basal, and 255.3%, 213.8%, 218.9% and 198.7% on Days 1, 2, 3 and 5. Maximum inhibition was observed 20 min after addition of the peptide on the day of control, after 5 min on Days 1, 2 and 3, and after 10 min on Day 5. Inhibition at the 5-min time point was 33.1% on the day of control, and 34.7%, 53.6%, 53.3% and 36.2% on the different days during the training course. The observed increment in VIP effect adds to prior reported data about increased VIP secretion during the training course, and may indicate enhanced physiological significance of VIP during stress.

KINETICS OF MURINE HAEMOPOIETIC CELL PROLIFERATION IN DIFFUSION CHAMBERS

Murine bone marrow cells were cultured in cell impermeable diffusion chambers in the abdominal cavities of mice. The kinetics of granulocyte and macrophage formation were studied by stathmokinetic and autoradiographic techniques. During the period of most rapid growth of proliferative granulocytes, their generation time and its different phases were: tc∽ 8 hr, tG1∽ 1·5 hr, ts∽ 5·5 hr, tG2∽ 0·7 hr and tM∽ 0·25 hr.

Receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes are upregulated during prolonged strain and energy deficiency

VIP receptors on blood mononuclear leucocytes and plasma VIP concentrations were studied during a ranger training course lasting for five days with almost continuous physical activity, and energy deficiency. The maximum binding capacity (Bmax) for the high affinity receptor increased (p less than 0.0005) from 0.71 (SEM = 0.11, N = 10) fmol/million cells to a maximum of 7.33 (SEM = 1.0) fmol/million cells on Day 4. There was no significant change in the dissociation constant (Kd) for the high affinity receptor, and no effect on Kd nor Bmax for the low affinity VIP receptor was detected. Plasma VIP concentration increased (p less than 0.0005) from 8.8 pmol/l (SEM = 0.6) to a maximum of 23.4 (SEM = 1.9) on the second day of the course. However, the highest plasma concentrations were about one order of magnitude lower than the dissociation constant (Kd) for the high affinity VIP receptor on the mononuclear leucocytes. These data indicate that heterologous upregulation of the high affinity VIP receptor on mononuclear blood cells takes place during combined strenuous physical exercise, and calorie deficiency.

REGULATION OF BONE MARROW CELL GROWTH IN DIFFUSION CHAMBERS: THE EFFECT OF GRANULOCYTE EXTRACTS

Hypotonic lysis of mature human blood granulocytes yielded an extract which reduced granulopoiesis and enhanced macrophage formation of mouse bone marrow cells cultured for 7 days in diffusion chambers (DC). The low molecular weight fraction (MW < 15,000–25,000 Daltons) obtained by Amicon filtration of the extract, reduced granulopoiesis without affecting macrophage formation. The high molecular weight fraction (MW > 15,000–25,000 Daltons) reduced the number of granulocytes and increased the number macrophages. Erythrocyte extract increased the macrophage formation in DC but did not alter the number of granulocytes. The spleen colony assay showed that the granulocyte extract increased the number of CFU‐S in DC. It is suggested that the granulocyte extract contain an inhibitor of stem cell differentiation to myeloid cells thereby reducing the number of proliferative granulocytes in DC 7 days later. The inhibitor of differentiation may lead to an increased self renewal of the stem cell in the DC system.

Colony inhibiting factor in mature granulocytes from normal individuals and patients with chronic myeloid leukemia

Inhibitory activity in extract from human blood granulocytes was tested on gran‐ulocyte‐macrophage colony formation in vitro. The inhibition depended on the type of serum used. With mouse BMC and FCS in the cultures, extract corresponding to 2.5 times 104 granulocytes/ml reduced the colony number by 35%, and extract from 2 times 105 cells caused maximal inhibition (80–90%). With HS and mouse BMC the colony number was reduced by only 11–12%, but stronger inhibition (55%) was observed when the serum concentration was reduced. With both types of sera the total cell number per culture plate was reduced relatively more than the colony number. Human GM‐CFC were as sensitive as mouse GM‐CFC, and extract from CML granulocytes inhibited less (p < 0.01) than extract from normal cells. Biochemical studies indicated that the inhibitor is a protein with a molecular weight of 30–60000. Lactoferrin, a putative inhibitor of CSF production, did not inhibit spontaneous or CSF‐induced colony formation in these studies.

Thymidine-dependent effect of granulocyte-derived inhibitor on granulocyte-macrophage progenitor cells (GM-CFC)

We have compared the effect of granulocyte extract (GRE) on proliferation of hemopoietic cells from various sources, using two different cultures media, CMRL 1066 and McCoy 5A. GRE caused a strong (80–90%) inhibition of granulocyte‐macrophage colony forming cells (GM‐CFC) in cultures with medium CMRL 1066. GM‐CFC in human blood and human and mouse bone marrow were equally sensitive to the inhibitor. The inhibitor had a maximal effect in the concentration range corresponding to GRE from 2 × 105 to 2 × 106 cells per 1 ml culture dish. At higher GRE concentration the inhibition was reduced. GM‐CFC from human blood and mouse marrow were suppressed in cultures with McCoys medium as well, but to a lesser extent than in CMRL 1066 cultures. On the other hand, in cultures with human bone marrow cells (BMC) and McCoys medium, GRE had no inhibitory effect. CMRL 1066 medium contains a number of components not present in McCoys medium. In a systematic study where these substances were added one by one to McCoys medium we found that inhibition by GRE depended upon the presence of thymidine. At a thymidine concentration of 3 × 10−5 mol/l GRE strongly suppressed GM‐CFC in human blood and bone marrow. This thymidine concentration itself had no effect. Other nucleosides or components of the CMRL 1066 did not potentiate the suppressive effect of GRE.

Cytotoxicity and effects of T2-toxin on plasma proteines involved in coagulation, fibrinolysis and kallikrein-kinin system

The activity of both the coagulation and fibrinolytic systems was markedly depressed 24 h after a sublethal dose of T-2 toxin. T-2 toxin was active as an anticoagulant at low doses, which did not affect the basal state of the animals. The kallikrein-kinin system was also affected by depletion of the prekallikrein, which indicates increased bradykinin levels in plasma. At the same time there was an increased activity of some clinically relevant enzymes in serum, indicating tissue injuries caused by T-2 toxin. All effects observed in this study reached their maximum within 24 h after administration, which corresponds to the time animals usually die when receiving a lethal dose. T-2 toxin does not, however, seem to affect the protease enzymes by reduced protein synthesis, because of early onset of the effects, nor does it act as a trigger itself. The effect of T-2 toxin on plasma protease enzymes is probably secondary to cytotoxic effects in the vascular endothelium.